Figure 6A Transverse section from a confocal z-stack showing the whole neural tube of a utr-mCherry embryo, showing localisation to the basal surface. B Transverse sections from a confocal z-stack of a non-apical progenitor labelled with a membrane marker in a utr-mCherry embryo to identify the basal surface of the spinal cord. Image sequence from confocal time-lapse shows the non-apical progenitor (-44m) undergoing mitosis to produce two neurons (-22m), of which one is not in contact with the basal surface. Both neurons extend nascent axons (0m). Data information: All scale bars = 5 µm.C Transverse sections from a confocal z-stack of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr-mCherry embryo to identify the basal surface of the spinal cord. Three different sections show the middle of the soma, the axon initiation site, and the axon tip. Green and magenta peaks in graphs show relative positions of cell membrane and basal surface, respectively. D Transverse sections from confocal z-stacks of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr-mCherry embryo injected with lamMO. Three different sections show the middle of the soma, the axon initiation site, and the axon tip. Green and magenta peaks in graphs show relative positions of cell membrane and basal surface, respectively. E Graphs showing distance (µm) between the basal edge of the soma, the axon initiation site or the axon tip and the basal surface of the spinal cord. Measurements were performed by measuring between basal-most green and magenta peaks in graphs of relative grey values (C, D). Bars show mean and standard deviation. WT: n = 5 cells from one experiment; lamMO: n = 11 cells from two experiments. Soma: WT mean = 2.007 µm, s.d. = 1.055; lamMO mean = 1.871 µm, s.d. = 1.830. Student's two-tailed test, P = 0.880. Axon initiation site: WT mean = 1.366 µm, s.d. = 3.13; lamMO mean = 2.353 µm, s.d. = 2.125. Student's two-tailed test, P = 0.468. Axon tip: WT mean = 0.637 µm, s.d. = 1.078; lamMO mean = 2.903 µm, s.d. = 2.0433. Student's two-tailed test, P = 0.037. Data information: All scale bars = 5 µm. * P < 0.05.F Image sequence from confocal time lapse shows a neuron in a Sly-/- embryo labelled with membrane and centrosome markers before (-40m), during (0) and after axon initiation (20m). Images are transverse reconstructions from confocal z-stacks.G Plots showing axon position on the soma relative to the cell centroid at 0,0 for dorsal and transverse view in Sly-/- embryos (n = 18 cells from three experiments). Axon position is not random (dorsal view P < 0.001, mean = 98.8o; transverse view P < 0.001, mean = 161.1o; Moore's modification of Rayleigh's test). H Plots showing merge of WT and Sly-/- axon positions on the cell body relative to cell centroid at 0,0 for dorsal and transverse views. Axon positions in WT and Sly-/- are not significantly different in dorsal view (0.2 < P < 0.5) but are in transverse view (P < 0.001; Batschelet's alternative to Hotelling test). WT: n = 86 cells from eight experiments; Sly-/-: n = 18 cells from three experiments.I Graph showing distance between centrosome and base of axon at time of axon initiation in WT and Sly-/- embryos. Bars show mean and standard deviation. WT: n = 26 cells from three experiments, mean = 10.13 µm, s.d. = 3.35. Sly-/-: n = 15 cells from two experiments, mean = 12.41 µm, s.d. = 3.281. One-way ANOVA, P = 0.123.J Plots showing centrosome position relative to the cell centroid at 0,0 for dorsal and transverse view in Sly-/- embryos (n = 15 cells from two experiments). Centrosome position is not random (dorsal view P < 0.001, mean = -129.0o; transverse view 0.001 < P < 0.005, mean = -57.0o; Moore's modification of Rayleigh's test). K Plots showing merge of WT and Sly-/- centrosome positions on the cell body relative to cell centroid at 0,0 for dorsal and transverse views. Centrosome positions are not significantly different between WT and Sly-/- (dorsal view 0.2 < P < 0.2, transverse view 0.1 < P < 0.2; Batschelet's alternative to Hotelling test). WT: n = 26 cells from three experiments; Sly-/-: n = 15 cells from two experiments.L Plots showing the positions of the centrosome and base of the axon in Sly-/- embryos at the time of axon initiation relative to the cell centroid at 0,0 for dorsal and transverse view (n = 15 cells from two experiments). Left-hand plots: centrosome position is not random (dorsal view P < 0.001, mean = -129.0o; transverse view P < 0.001, mean = -57.0) and axon position is not random (dorsal view P < 0.001, mean = 95.1o; transverse view P < 0.001, mean = 168.8o, Moore's modification of Rayleigh's test). Centrosome and axon positions are significantly different (dorsal view 0.001 > P; transverse view 0.001 > P; Moore's test for paired data). Right-hand plots: vectors connecting centrosome and nascent axon from the same cell are not random (dorsal view P < 0.001, mean = 66.1o; transverse view P < 0.001, mean = 150.9o).